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[
International Worm Meeting,
2015]
A pair of ASE chemosensory neurons, ASEL and ASER, are major salt sensors, and play critical roles in chemotaxis to NaCl. Calcium imaging has previously revealed that ASEL and ASER are activated by an increase and decrease in NaCl concentrations, respectively (Suzuki et al., 2008; Ortiz et al., 2009). These asymmetric responses by ASEL and ASER to changes of NaCl concentrations are crucial to efficient chemotaxis of C. elegans toward higher concentrations of NaCl. While Goodman et al. (1998) reported in situ whole-cell patch-clump recording of ASER, electrophysiological characterisation of ASE neurons is still required to understand how the neurons respond to the NaCl concentration changes.Toward the goal, we have investigated electrophysiological properties of ASE neurons in wild-type C. elegans by in vivo whole-cell patch-clamp recordings, and have found that both of ASE neurons showed resting membrane potentials of approximately -60 mV and membrane resistances of about 2 Gomega. In both of ASE neurons, voltage responses to current injections showed solitary action potentials. Depolarization of wild-type ASEL was observed when a puff of 150 mM NaCl was applied to the animal's nose in bath solution containing 50 mM NaCl. On the other hand, a puff of NaCl-free buffer induced ASER depolarization. These results are consistent with those of calcium imaging. To understand roles of the action potentials in ASE, we are currently trying to analyse electrophysiological properties of ASE neurons in various mutants.References1. Suzuki et al., Nature 454: 114-118 (2008)2. Ortiz et al., Current Biology 19: 996-1004 (2009)3. Goodman et al., Neuron 20: 763-772 (1998).
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[
International Worm Meeting,
2003]
Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991
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[
J Mol Neurosci,
2006]
Neurotransmitter-gated receptors are assembled in the endoplasmic reticulum and transported to the cell surface through a process that might be of central importance to regulate the efficacy of synaptic transmission (Kneussel and Betz, 2000; Kittler and Moss, 2003). This process is relatively inefficient- what may be the consequence of tight quality controls that guarantee the functional competence of the final product. For this purpose, specific proteins involved in assembly and trafficking of receptors might be required (Keller and Taylor, 1999; Millar, 2003; Wanamaker et al., 2003). The RIC-3 protein could be one of them, as mutations in the
ric-3 gene affect maturation of nicotinic acetylcholine receptors (nAChRs) in Caenorhabditis elegans (Halevi et al., 2002). Moreover, the human homolog hRIC-3 showed differential effects when coexpressed with several ligand-gated receptors (Halevi et al., 2003). Thus, it enhanced alpha7 nAChR expression while inhibiting expression of other nAChR subtypes (alpha4beta2 and alpha3beta4) and 5-HT3 serotonin receptors (5-HT3Rs). These opposite effects suggested that the RIC-3 protein might play a key role in the biogenesis of some ligand-gated receptors and prompted us to investigate how it performs its action. Here, we show that the RIC-3 protein acts as a barrier for some receptors like alpha4beta2 nAChRs and 5-HT3Rs, stopping the traffic of mature receptors to the membrane. In contrast, the inefficient transport of alpha7 nAChRs is enhanced by RIC-3 in a process in which certain amino acids at the amphipathic helix located at the C-terminal region of the large cytoplasmic domain are involved.
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[
J Cell Biol,
1988]
The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.
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[
J Cell Biol,
1985]
Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D.M. III, I. Ortiz, G.C. Berliner, and H.F. Epstein, 1983, Cell, 34: 477-490). Biochemical and electron microscope studies of KCl-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.
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[
MicroPubl Biol,
2021]
Parasitic nematode infections continue to have an enormous impact on human and livestock health worldwide (Hotez et al., 2014; Kaplan & Vidyashankar, 2012). A limited arsenal of anthelmintic drugs are available to combat these infections. One of the most widely used classes is benzimidazoles (BZ), and resistance against this class is widespread (Kaplan & Vidyashankar, 2012). Previous studies to understand parasitic nematode resistance using the free-living model organism Caenorhabditis elegans showed that variation in the C. elegans beta-tubulin gene
ben-1, an ortholog of beta-tubulins in parasitic nematodes, confers resistance to BZ drugs (Dilks et al., 2020; Driscoll et al., 1989; Hahnel et al., 2018). The most common missense mutation resistance alleles are F167Y, E198A, and F200Y (Avramenko et al., 2019; Mohammedsalih et al., 2020). Although computational models have predicted that these amino acids are involved in the binding of BZ compounds to beta-tubulins, the binding remains to be investigated empirically at the structural level because nematode-specific beta-tubulin structures have not been created (Aguayo-Ortiz et al., 2013; Hahnel et al., 2018). To better understand the mechanisms of resistance, we sought to obtain those crystallographic structures.
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[
International Worm Meeting,
2009]
Chemosensation allows animals to evaluate their environment, detect food, other animals, and dangerous toxins while responding with appropriate behaviors essential to the animal''s survival. A robust chemosensory system can be generated even from a seemingly simple nervous system, such as that of C. elegans, which can detect and respond to a vast number of chemical cues. One important, but poorly understood, strategy used by C. elegans is to "lateralize" the function of some of its sensory neurons, such as the ASE neurons, thus increasing the discriminatory power of a system comprised of relatively few elements. We have found that the ASE neurons respond to several salt cues and these responses are asymmetric in terms of whether the left or the right ASE neuron responds to a specific salt cue (Ortiz et al., submitted). Previous work in our lab has furthermore identified a number of guanylyl cyclase genes as having a role in the chemotaxis asymmetry of ASE (Ortiz et al., submitted). Mutant analysis has revealed that individual gcy genes are specifically required for sensing particular ions. Such specificity could be conferred through either the protein''s receptor family ligand-binding region (RFLBR) in its extracellular domain or by the protein''s guanylyl cyclase (GC) domain in its intracellular region. We seek to identify the molecular mechanisms by which these asymmetrically expressed receptor type guanylyl cyclases confer the specificity that underlies this lateralization. In order to test these predictions, intra- or extra-cellular domains of individual GCY proteins were swapped, chimeric proteins were introduced into mutant background animals in a cell-specific manner, and then rescue of chemotaxis defects were tested. The individual GCY protein domains that confer the cellular specificity of ASE neurons, which enables them to mediate responses only to particular cues are identified by evaluating assay output. Results from such experiments allow for the characterization of the domain(s) essential for specificity. By identifying these molecular mechanisms, key predictions of the role that these proteins play in ASE neurons, putatively functioning either as chemoreceptors or, alternatively, as signal transducers, can begin to be tested. We will pursue further strategies for elucidating these molecular mechanisms as part of an overall effort in exploring the relationship between individual genes, their patterns of expression in specific cellular contexts, and the chemosensory behaviors exhibited by C. elegans in response to salt cues found in its environment.
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[
Midwest Worm Meeting,
2000]
O2 homeostasis is essential for the survival of all organisms. From bacteria to mammals, cellular O2 levels are carefully regulated. Low O2 (hypoxia) will endanger ATP generation by shutting down the oxidative phosphorylation. In mammals hypoxia-inducible factor (HIF-1) plays a central role in regulation of responses to low O2 levels. It consists of two bHLH-PAS transcriptional subunits, HIF-1a and HIF-1b. HIF-1b is better known as aryl hydrocarbon receptor nuclear translocator (ARNT). Under hypoxic conditions, HIF-1a protein is stabilized and dimerizes with ARNT to form an active transcription heterodimer (HIF-1), which in turn activates the transcription of downstream targets. The target gene products either promote O2 delivery or switch metabolism to low O2 consumption. Multiple mechanisms of O2 sensing have been proposed, but none have been vigorously tested in vivo (1). Here, we report that bHLH-PAS genes may mediate response to hypoxia in C. elegans. The C. elegans genome encodes 5 putative transcription factors that contain basic-helix-loop-helix and PAS domain motifs. Two of these genes,
ahr-1 and
aha-1, encode the orthologs of the aryl hydrocarbon receptor and ARNT, respectively (2). Genetic analysis of these two genes has demonstrated that
aha-1 is essential for viability, while
ahr-1 is not (3,4). This indicates that AHA-1 may dimerize with other bHLH-PAS proteins to regulate developmentally important processes. Four lines of evidence suggest that AHA-1 and F38A6.3a may form hypoxia-responsive complex in C. elegans. First, AHA-1 and the F38A6.3a gene product can be co-immunoprecipitated when expressed in vitro. Second, F38A6.3a mRNA levels increase in hypoxic conditions. Third, we have identified potential target genes that are induced by hypoxia in N2, but not in animals homozygous for a deletion in F38A6.3a. Fourth, AHA-1 is expressed in many tissues (3, 4), and F38A6.3a:GFP is ubiquitously expressed. Further characterization of the mutant phenotype will be presented at the meeting. References: 1. Semenza GL. Perspectives on Oxygen Sensing. Cell (1999) 98(3): 281-4. 2. Powell-Coffman JA, Bradfield CA, Wood WB. Caenorhabditis elegans orthologs of the aryl hydrocarbon receptor and its heterodimerization partner with the aryl hydrocarbon receptor nuclear translocator. PNAS (1998), 95: 2844-2849. 3. Jiang H, Guo R, Powell-Coffman JA. An essential role for
aha-1, a PAS-domain-containing regulatory gene in C. elegans. 1999 International Worm Meeting, 438. 4. See other Powell-Coffman lab abstracts for this meeting.
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[
East Coast Worm Meeting,
1998]
The Hedgehog family of signaling molecules controls many aspects of animal development, from embryonic patterning to limb formation. Recently, these proteins have been shown to rely on a novel mechanism to regulate their spatial distribution (1,2). Hedgehog proteins undergo an autoprocessing reaction to yield two distinct products, an amino-terminal fragment (Hh-N), and a carboxy-terminal fragment (Hh-C). Hh-N functions in signaling, whereas Hh-C mediates the processing reaction. This reaction proceeds via an internal thioester intermediate and results in the covalent linkage of cholesterol to Hh-N. This modification in turn causes Hh-N to remain tightly associated with the cell surface, thus effectively limiting its free diffusion and range of action. We are interested in determining whether similar mechanisms may be operating in the biogenesis of other secreted molecules. Towards this goal, we have started to characterize a set of eight C. elegans proteins with sequence similarity to the Hh-C domain of Hedgehog (2,3). In these proteins, as in the Hedgehog family, the Hh-C-like domain is located in the carboxy-teminal end, and is preceded by an amino-terminal domain bearing a signal sequence (but with otherwise no homology to Hh-N). This structure is consistent with the possibility that these proteins are secreted and undergo processing in a manner similar to that described for Hedgehog. To test this possibility we have fused the Hh-C-like domain of one of these proteins to a His tag, and purified the fusion protein from E. coli. We find that such a fusion can undergo the processing reaction in vitro, suggesting that the Hh-C-like domain is functionally active in the nematode proteins. We have begun to characterize the functions of Hh-C-related proteins using RNA-mediated interference. Our initial results suggest that one of these proteins, T05C12, is essential for molting. Injection of T05C12 double-stranded RNA results in 90% larval lethality. The larvae apparently die from a failure to shed old cuticles during molts. In many animals, a cuticular plug can be seen obstructing the mouth opening where it presumably interferes with feeding. The amino-terminal domain of T05C12 contains several sequence motifs similar to those found in collagens and other extracellular matrix proteins, raising the possibility that the T05C12 product is a cuticle component. Consistent with this possibility, a T05C12:GFP fusion is expressed in hypodermal cells during larval and adult stages. We are currently determining the effects of expressing mutant forms of T05C12 to test the function of its Hh-C-like domain. 1. Porter, JA. et al. (1996). Cell 86, 21-34. 2. Porter, JA. et al. (1996). Science 274, 255-259. 3. Burglin, TR. (1996). Current Biology 6, 1047-1050.
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[
J Cell Biol,
1996]
Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.